Endothelial Surface Layer Research Articles

Rebuttal from David C. Poole.

Direct observation of the muscle microcirculation at rest reveals moving red blood cells (RBCs) in most capillaries, and this is consonant with dye injections in conscious animals and rest and exercise measurements of muscle [hemoglobin] in humans (Fraser et al. 2012; Poole, 2014; Poole et al. 2011, 2013; Richardson et al. 2003). There is substantial heterogeneity of RBC flux and haematocrit among capillaries and across time within capillaries. Thus, the notion of a given capillary, or unit of its surface area, representing a quantum unit of exchange either for RBC-borne O2 or for plasma substrates is absurd. The presence of the endothelial surface layer (glycocalyx) means that RBCs and plasma move at very different rates. This phenomenon renders the notion of ‘blood volume’ obscure because the volumetric proportion of RBCs versus plasma varies widely and is dependent upon flow rate. My learned colleagues (Barrett et al. 2014) find evidence for insulin-induced capillary recruitment in skin (Meijer et al. 2012). Whilst suggesting that, for some undisclosed reason, thin muscles are not representative of thicker muscles, they suppose that skin capillaries behave synonymously to those in muscle, contrary to their own data (Meijer et al. 2012). Profound organ-dependent differences exist with respect to capillary structure and function (Poole, 2014; Poole et al. 2011, 2013). The support by Barrett et al. (2014) for de novo capillary recruitment is predicated upon model-based interpretation of increased contrast-enhanced ultrasound bubbles and 1-methyl xanthine metabolism with insulin (non-flow dependent) and muscle contractions (partly flow dependent). These indirect techniques have been heavily criticized (Poole, 2014, Poole et al. 2011, 2013). Moreover, their observed microvascular volume increases are accounted for in toto by modification of the endothelial surface layer (Eskens et al. 2013). In addition, the studies by Barrett and colleagues (Inyard et al. 2007; Barrett et al. 2014) are implausible because contractions in healthy muscle were not accompanied by an increased blood flow‡ The wrong view of science obscures basic mechanisms of function/ dysfunction. For instance, heart failure and diabetes (type I/II) decrease the proportion of muscle capillaries supporting flow (Poole et al. 2011, 2012, 2013). An understanding of this phenomenon helps to explain the reduced O2 diffusing capacity and impaired glucose uptake in these diseases. This pathology would be undetected if massive capillary stasis were considered normal, and the correct therapeutic targets may be missed. In closing, Barrett et al. (2014) and I agree that ‘microvascular recruitment’ is important for increased delivery of O2 and substrates to muscle. However, the nature of that recruitment is not massive initiation of RBC flux in non-flowing vessels with insulin or contractions but, rather, an expansion of capillary volume by endothelial surface layer modification and longitudinal recruitment of exchange capacity along the length of already flowing capillaries.

Glomerular endothelial cell injury and cross talk in diabetic kidney disease.

Diabetic kidney disease (DKD) remains a leading cause of new-onset end-stage renal disease (ESRD), and yet, at present, the treatment is still very limited. A better understanding of the pathogenesis of DKD is therefore necessary to develop more effective therapies. Increasing evidence suggests that glomerular endothelial cell (GEC) injury plays a major role in the development and progression of DKD. Alteration of the glomerular endothelial cell surface layer, including its major component, glycocalyx, is a leading cause of microalbuminuria observed in early DKD. Many studies suggest a presence of cross talk between glomerular cells, such as between GEC and mesangial cells or GEC and podocytes. PDGFB/PDGFRβ is a major mediator for GEC and mesangial cell cross talk, while vascular endothelial growth factor (VEGF), angiopoietins, and endothelin-1 are the major mediators for GEC and podocyte communication. In DKD, GEC injury may lead to podocyte damage, while podocyte loss further exacerbates GEC injury, forming a vicious cycle. Therefore, GEC injury may predispose to albuminuria in diabetes either directly or indirectly by communication with neighboring podocytes and mesangial cells via secreted mediators. Identification of novel mediators of glomerular cell cross talk, such as microRNAs, will lead to a better understanding of the pathogenesis of DKD. Targeting these mediators may be a novel approach to develop more effective therapy for DKD.

Numerical analysis for elucidation of nonlinear frictional characteristics of a deformed erythrocyte moving on a plate in medium subject to inclined centrifugal force.

Complex interactions between blood cells, plasma proteins, and glycocalyx in the endothelial surface layer are crucial in microcirculation. To obtain measurement data of such interactions, we have previously performed experiments using an inclined centrifuge microscope, which revealed that the nonlinear velocity-friction characteristics of erythrocytes moving on an endothelia-cultured glass plate in medium under inclined centrifugal force are much larger than those on plain or material-coated glass plates. The purpose of this study was to elucidate the nonlinear frictional characteristics of an erythrocyte on plain or material-coated glass plates as the basis to clarify the interaction between the erythrocyte and the endothelial cells. We propose a model in which steady motion of the cell is realized as an equilibrium state of the force and moment due to inclined centrifugal force and hydrodynamic flow force acting on the cell. Other electrochemical effects on the surfaces of the erythrocyte and the plate are ignored for the sake of simplicity. Numerical analysis was performed for a three-dimensional flow of a mixture of plasma and saline around a rigid erythrocyte model of an undeformed biconcave shape and a deformed shape with a concave top surface and a flat bottom surface. A variety of conditions for the concentration of plasma in a medium, the velocity of the cell, and the minimum gap width and the angle of attack of the cell from the plate, were examined to obtain the equilibrium states. A simple flat plate model based on the lubrication theory was also examined to elucidate the physical meaning of the model. The equilibrium angle of attack was obtained only for the deformed cell model and was represented as a power function of the minimum gap width. A simple flat plate model qualitatively explains the power function relation of the frictional characteristics, but it cannot explain the equilibrium relation, confirming the computational result that the deformation of the cell is necessary for the equilibrium. The frictional characteristics obtained from the present computation qualitatively agree with those of former experiments, showing the validity of the proposed model.

Role of the vascular wall in sodium homeostasis and salt sensitivity.

Excessive sodium intake is associated with both hypertension and an increased risk of cardiovascular events, presumably because of an increase in extracellular volume. The extent to which sodium intake affects extracellular volume and BP varies considerably among individuals, discriminating subjects who are salt-sensitive from those who are salt-resistant. Recent experiments have shown that, other than regulation by the kidney, sodium homeostasis is also regulated by negatively charged glycosaminoglycans in the skin interstitium, where sodium is bound to glycosaminoglycans without commensurate effects on extracellular volume. The endothelial surface layer is a dynamic layer on the luminal side of the endothelium that is in continuous exchange with flowing blood. Because negatively charged glycosaminoglycans are abundantly present in this layer, it may act as an intravascular buffer compartment that allows sodium to be transiently stored. This review focuses on the putative role of the endothelial surface layer as a contributor to salt sensitivity, the consequences of a perturbed endothelial surface layer on sodium homeostasis, and the endothelial surface layer as a possible target for the treatment of hypertension and an expanded extracellular volume.

Melanoma-derived IL-1 converts vascular endothelium to a proinflammatory and procoagulatory phenotype via NFκB activation.

Spreading of melanoma is associated with efficient extravasation of circulating tumor cells from the vascular system into distant target organs. This process is accompanied and supported by proinflammatory and procoagulatory conditions. In this study, we analysed the ability of human melanoma cell lines to activate endothelial cells (ECs) in vitro. Some melanoma cells, that is, MV3, were shown to trigger an prompt calcium-flux-dependent, procoagulatory endothelial response that was accompanied by luminal release of ultra-large von Willebrand factor (ULVWF) fibres that were immobilized to the endothelial surface layer. In contrast to MV3-derived supernatant, prolonged treatment of ECs with WM9-derived supernatant mediated a pronounced activation of nuclear factor kappa B (NFκB). NFκB activation in ECs was dependent on both IL-1α and IL-1β secreted from melanoma cells. Melanoma-derived IL-1 mediated an upregulation of proinflammatory cytokines IL-6 and IL-8, the intercellular adhesion molecule-1 (ICAM-1), the vascular cell adhesion molecule-1 (VCAM-1) and the procoagulatory tissue factor (TF) in ECs. Our data show that melanoma cells activate ECs either directly and within seconds or by an IL-1-mediated NFκB activation. Both pathways of EC activation convert the regular repressive function of ECs on inflammation and coagulation to a proinflammatory and procoagulatory surface that supports tumor progression.

Clinical use of hydroxyethyl starch and serious adverse effects: Need for awareness amongst the medical fraternity

Clinical use of hydroxyethyl starch and serious adverse effects: Need for awareness amongst the medical fraternity

Tackling scale in biomedical flows

Fluid flow problems in the areas of biomedical engineering and biomechanics are being addressed at increasingly small scales. This is very apparent in the domain of theoretical modelingwheremultiscalemodeling approaches are steadily on the rise. In addition to utilizing standard computational fluidmechanicsmodels at reduced scales, increasingly lattice Boltzmann and molecular dynamics models are employed to lower the level of these challenging scales even further. For example, multicoupled, multiscale modeling strategies are essential to successfully address the mechanobiologic problem of atherosclerotic plaque initiation at the vascular endothelium where particulates like blood cells as well as biomolecules are involved in the mechanotransductive processes across the endothelial surface layer into the underlying tissue. Similarly, in the field of experimental biofluid mechanics, the wealth of more recently developed imaging techniques and not least of ultrafine optical measurement techniques allow for investigation at the involved small length scales and have set the research bar notably higher of late. The idea for a special issue addressing scale issues in biomedical flows was seeded as a result of the 2011 Conference on Micro and Nano Flows (MNF2011), held in Thessaloniki, Greece, August 22–24, which was the third, suc-

Association of Kidney Function with Changes in the Endothelial Surface Layer

ESRD is accompanied by endothelial dysfunction. Because the endothelial glycocalyx (endothelial surface layer) governs interactions between flowing blood and the vessel wall, perturbation could influence disease progression. This study used a novel noninvasive sidestream-darkfield imaging method, which measures the accessibility of red blood cells to the endothelial surface layer in the microcirculation (perfused boundary region), to investigate whether renal function is associated with endothelial surface layer dimensions. Perfused boundary region was measured in control participants (n=10), patients with ESRD (n=23), participants with normal kidney function after successful living donor kidney transplantation (n=12), and patients who developed interstitial fibrosis/tubular atrophy after kidney transplantation (n=10). In addition, the endothelial activation marker angiopoietin-2 and shed endothelial surface layer components syndecan-1 and soluble thrombomodulin were measured using ELISA. Compared with healthy controls (1.82 ± 0.16 µm), ESRD patients had a larger perfused boundary region (+0.23; 95% confidence interval, 0.46 to <0.01; P<0.05), which signifies loss of endothelial surface layer dimensions. This large perfused boundary region was accompanied by higher circulating levels of syndecan-1 (+57.71; 95% confidence interval, 17.38 to 98.04; P<0.01) and soluble thrombomodulin (+12.88; 95% confidence interval, 0.29 to 25.46; P<0.001). After successful transplantation, the perfused boundary region was indistinguishable from healthy controls (without elevated levels of soluble thrombomodulin or syndecan-1). In contrast, however, patients who developed interstitial fibrosis and tubular atrophy showed a large perfused boundary region (+0.36; 95% confidence interval, 0.09 to 0.63; P<0.01) and higher levels of endothelial activation markers. In addition, a significant correlation between perfused boundary region, angiopoietin-2, and eGFR was observed (perfused boundary region versus GFR: Spearman's ρ=0.31; P<0.05; perfused boundary region versus angiopoietin-2: Spearman's ρ=-0.33; P<0.05). Reduced renal function is strongly associated with low endothelial surface layer dimensions. After successful kidney transplantation, the endothelial surface layer is indistinguishable from control.

TNF-mediated damage to glomerular endothelium is an important determinant of acute kidney injury in sepsis

Severe sepsis is often accompanied by acute kidney injury (AKI) and albuminuria. Here we studied whether the AKI and albuminuria associated with lipopolysaccharide (LPS) treatment in mice reflects impairment of the glomerular endothelium with its associated endothelial surface layer. LPS treatment decreased the abundance of endothelial surface layer heparan sulfate proteoglycans and sialic acid, and led to albuminuria likely reflecting altered glomerular filtration perm-selectivity. LPS treatment decreased the glomerular filtration rate (GFR), while also causing significant ultrastructural alterations in the glomerular endothelium. The density of glomerular endothelial cell fenestrae was 5-fold lower whereas the average fenestrae diameter was 3-fold higher in LPS-treated than in control mice. The effects of LPS on the glomerular endothelial surface layer, endothelial cell fenestrae, GFR, and albuminuria were diminished in TNF receptor 1 (TNFR1) knockout mice, suggesting that these LPS effects are mediated by TNF-α activation of TNFR1. Indeed, intravenous administration of TNF decreased GFR and led to loss of glomerular endothelial cell fenestrae, increased fenestrae diameter, and damage to the glomerular endothelial surface layer. LPS treatment decreased kidney expression of vascular endothelial growth factor (VEGF). Thus, our findings confirm the important role of glomerular endothelial injury, possibly by a decreased VEGF level, in the development and progression of AKI and albuminuria in the LPS model of sepsis in the mouse.

Injury to the endothelial surface layer induces glomerular hyperfiltration rats with early-stage diabetes.

Glomerular endothelial surface layer (ESL) may play a role in the mechanisms of albuminuria in diabetic nephropathy, which lack evidence in vivo. The effects of high glucose on the passage of albumin across the glomerular ESL were analysed in streptozotocin-induced diabetic Sprague-Dawley rats for 4 weeks. Albuminuria and glomerular mesangial matrix were significantly increased in diabetic rats. The passage of albumin across the ESL, as measured by albumin-colloid gold particle density in the glomerular basement membrane (GBM), was increased significantly in diabetic rats. The thickness of the glomerular ESL, examined indirectly by infusing Intralipid into vessels using an electron microscope, was significantly decreased and the GBM exhibited little change in diabetic rats. In summary, the glomerular ESL may play a role in the pathogenesis of albuminuria in rats with early-stage diabetes.

The endothelium as part of the integrative glomerular barrier complex

A new study by Xu et al. presents compelling evidence for an important role of the glomerular endothelium in acute kidney injury. They show that lipopolysaccharide reduces the endothelial surface layer, resulting in mild albuminuria, reduced glomerular filtration rate, and fewer endothelial fenestrae. Tumor necrosis factor-α (TNF-α) is identified as instrumental in these lipopolysaccharide effects through the TNF-α type 1 receptor. The study highlights that the glomerular endothelium has a key role in the maintenance of the glomerular filtration barrier.

G protein-coupled receptors mediate coronary flow- and agonist-induced responses via lectin-oligosaccharide interactions

Blood flow acts parallel to the coronary luminal endothelial surface layer (LESL) and modulates multiple parenchymal functions via the release of paracrine agents. Evidence indicates that the LESL may be a flow-sensing organelle and that perhaps through flow-induced lectin (L)·oligosaccharide (O) complex formation (L·O) participates in this process. LESL integrins and selectins are both lectinic and flow sensitive, but the L properties of flow-sensitive G protein-coupled receptors (GPCRs) are unknown. Therefore, we investigated the presence of L in the LESL and hypothesized that if flow-sensitive GPCRs are L, flow and O will determine their response to receptor activation. The LESL protein fraction isolated from guinea pig hearts was passed through an affinity chromatography column made of three sugars, mannose, galactose, and N-acetylglucosamine, and the lectinic fraction was eluted. Immune dot blot was used to identify L proteins in the LESL fraction. Our results indicate the following. 1) Two-dimensional SDS-PAGE (2D-SDS-PAGE) of the LESL lectinic fraction revealed at least 167 Ls. 2) Among these Ls, we identified three selectins and the GPCRs: angiotensin II, bradykinin (B2-R), adenosine A1 and A2, prolactin, endothelin, α1-adrenergic (α1A-R), thromboxane A2, β1-adrenergic, β3-adrenergic, and insulin receptors; the first six GPCRs are known to be flow sensitive. 3) The amplitude of receptor-induced vascular responses by α1A-R and B2-R activation (phenylephrine or bradykinin, respectively) was a function of flow and O (hyaluronidate). Our results support a novel mechanism of GPCR-mediated responses to flow via L·O interaction.

The role of the glomerular endothelium in albumin handling.

The unique permeability characteristics of the glomerular capillary wall depend on its three-layer structure, consisting of endothelial cells, the basement membrane and podocytes. These components form the glomerular filtration barrier (GFB). That albuminuria may occur in the absence of changes in podocyte foot processes suggests that GFB components other than podocytes have essential roles in albumin handling. The endothelium forms the first part of the GFB and is characterized by fenestrations-transcellular holes that are filled with endothelial glycocalyx, a hydrated mesh principally comprised of proteoglycans. The glycocalyx and adsorbed plasma constituents form the endothelial surface layer (ESL). Human and animal studies have shown that the glomerular ESL restricts macromolecule passage and ensures that plasma albumin is largely excluded from the GFB. The glomerular endothelium is also likely to indirectly influence glomerular albumin handling by modifying podocyte behaviour. These modifications may occur physiologically through soluble mediators and/or pathologically through increased exposure of podocytes to plasma components as a consequence of ESL dysfunction. The importance of the glomerular endothelium and ESL in albumin handling also sheds light on the relationship between albuminuria and vascular disease. The therapeutic potential that this relationship offers will become evident with better understanding of the structure, composition and regulation of the glycocalyx.

Reappraising Starling

Vascular permeability is traditionally explained by Starling's principle, describing two opposing forces across the endothelial cell line to maintain compartments in balance. Several contradictions to this principle have recently questioned its validity. Hydraulic conductivity is kept low by a properly working endothelial surface layer, created by binding and intercalating plasma constituents with the structural elements of an endothelial glycocalyx. Limiting fluid filtration is not closely related to the interstitial protein concentration. Rather, the oncotic pressure difference pertinent to fluid homeostasis is built up between the intravascular space and a small protein-free zone beneath the protein-loaded endothelial glycocalyx. This crucial structure, and therefore the resistance of the barrier against outflow of large molecules, is endangered by ischaemia, inflammation and intravascular hypervolaemia. An intact endothelial surface layer retains iso-oncotic preparations of large molecules infused to compensate for acute bleeding. Crystalloids cannot be held back sufficiently, even if preload is warranted. Starling's principle requires an adaptation to recognize that there is no inward-directed oncotic pressure gradient across the whole anatomical vessel wall. The carrier of vascular barrier competence is the intact endothelial surface layer which might be protected by avoiding intravascular hypervolaemia and limiting inflammation.

Correction: Nanomechanics and Sodium Permeability of Endothelial Surface Layer Modulated by Hawthorn Extract WS 1442

Correction: Nanomechanics and Sodium Permeability of Endothelial Surface Layer Modulated by Hawthorn Extract WS 1442

Plane Couette-Poiseuille flow past a homogeneous poroelastic layer

An analytical solution is presented for the problem of fully developed plane Couette-Poiseuille flow past a homogeneous, permeable poroelastic layer. Main novel feature of this work is that the compressibility, which is related to the action of the free-fluid pressure on the poroelastic layer, is taken into account. Therefore, the solid stress problem is two-dimensional, although the fluid flow problem remains one-dimensional in the limit of infinitesimal strain. The pressure-related compressibility affects strongly the distribution of the von Mises stress in the poroelastic layer by shifting the local maximum towards the high-pressure region of the channel. Furthermore, the established analytical solution is used to investigate some aspects of the mechanotransducing role of the endothelial surface layer. A compressible surface layer might serve as a “bumper’’ by reducing the magnitude of the overall shearing force (viscous and elastic) acting on endothelial cells, as compared to the magnitude of the fluid shear force that would be exerted in its absence.

Glycosaminoglycan Regulation by VEGFA and VEGFC of the Glomerular Microvascular Endothelial Cell Glycocalyx in Vitro

Damage to endothelial glycocalyx impairs vascular barrier function and may contribute to progression of chronic vascular disease. An early indicator is microalbuminuria resulting from glomerular filtration barrier damage. We investigated the contributions of hyaluronic acid (HA) and chondroitin sulfate (CS) to glomerular microvascular endothelial cell (GEnC) glycocalyx and examined whether these are modified by vascular endothelial growth factors A and C (VEGFA and VEGFC). HA and CS were imaged on GEnCs and their resynthesis was examined. The effect of HA and CS on transendothelial electrical resistance (TEER) and labeled albumin flux across monolayers was assessed. Effects of VEGFA and VEGFC on production and charge characteristics of glycosaminoglycan (GAG) were examined via metabolic labeling and liquid chromatography. GAG shedding was quantified using Alcian Blue. NDST2 expression was examined using real-time PCR. GEnCs expressed HA and CS in the glycocalyx. CS contributed to the barrier to both ion (TEER) and protein flux across the monolayer; HA had only a limited effect. VEGFC promoted HA synthesis and increased the charge density of synthesized GAGs. In contrast, VEGFA induced shedding of charged GAGs. CS plays a role in restriction of macromolecular flux across GEnC monolayers, and VEGFA and VEGFC differentially regulate synthesis, charge, and shedding of GAGs in GEnCs. These observations have important implications for endothelial barrier regulation in glomerular and other microvascular beds.

Localization of fluorescent particles with nanometer precision using standard microscopy and a point spread function approach

BackgroundResolution of optical microscopy is limited by wave optics to about 200 to 400 nm. However, diffraction patterns generated by individual fluorescent particles (point spread functions, PSF) contain location information with much higher precision. Here, we describe a user‐friendly approach for single fluorescence particle 3D localization and tracking with nanometer precision in a standard fluorescence microscope.MethodsImages of fluorescent particles were obtained over a large range (about 7 μm) of vertical positions and analysed by whole pattern (WP) comparison with a PSF recorded for the specific setup and measurement of the outermost ring radius (ORR).ResultsFor a vertical range of 4.5 μm, standard deviation (SD) from the predicted value, indicating validity, was 15.6 and 23.5 nm for WP and ORR. Underexposure or overlapping images led only to a slight increase in SD. The precision, determined as SD of the distance between two particles, ranged from 3 to 6 nm. Local viscosity may be determined from random particle movement with a precision of about 5% of the measured value.ConclusionWith a validity in the range of 20 nm and a precision of about 4–5 nm obtained by standard fluorescent microscopy, the PSF approach offers a valuable tool for a variety of experimental investigations of particle/structure localizations and local viscosity, e.g. in studies of the endothelial surface layer.

Glomerular Endothelial Surface Layer Acts as a Barrier against Albumin Filtration

Glomerular endothelium is highly fenestrated, and its contribution to glomerular barrier function is the subject of debate. In recent years, a polysaccharide-rich endothelial surface layer (ESL) has been postulated to act as a filtration barrier for large molecules, such as albumin. To test this hypothesis, we disturbed the ESL in C57Bl/6 mice using long-term hyaluronidase infusion for 4 weeks and monitored albumin passage using immunolabeling and correlative light-electron microscopy that allows for complete and integral assessment of glomerular albumin passage. ESL ultrastructure was visualized by transmission electron microscopy using cupromeronic blue and by localization of ESL binding lectins using confocal microscopy. We demonstrate that glomerular fenestrae are filled with dense negatively charged polysaccharide structures that are largely removed in the presence of circulating hyaluronidase, leaving the polysaccharide surfaces of other glomerular cells intact. Both retention of native ferritin [corrected] in the glomerular basement membrane and systemic blood pressure were unaltered. Enzyme treatment, however, induced albumin passage across the endothelium in 90% of glomeruli, whereas this could not be observed in controls. Yet, there was no net albuminuria due to binding and uptake of filtered albumin by the podocytes and parietal epithelium. ESL structure and function completely recovered within 4 weeks on cessation of hyaluronidase infusion. Thus, the polyanionic ESL component, hyaluronan, is a key component of the glomerular endothelial protein permeability barrier.

&lt;em&gt;In vivo&lt;/em&gt; Measurement of the Mouse Pulmonary Endothelial Surface Layer

The endothelial glycocalyx is a layer of proteoglycans and associated glycosaminoglycans lining the vascular lumen. In vivo, the glycocalyx is highly hydrated, forming a substantial endothelial surface layer (ESL) that contributes to the maintenance of endothelial function. As the endothelial glycocalyx is often aberrant in vitro and is lost during standard tissue fixation techniques, study of the ESL requires use of intravital microscopy. To best approximate the complex physiology of the alveolar microvasculature, pulmonary intravital imaging is ideally performed on a freely-moving lung. These preparations, however, typically suffer from extensive motion artifact. We demonstrate how closed-chest intravital microscopy of a freely-moving mouse lung can be used to measure glycocalyx integrity via ESL exclusion of fluorescently-labeled high molecular weight dextrans from the endothelial surface. This non-recovery surgical technique, which requires simultaneous brightfield and fluorescent imaging of the mouse lung, allows for longitudinal observation of the subpleural microvasculature without evidence of inducing confounding lung injury.